1. Field of the Invention
The present invention relates to methods and materials for targeting and enhancing RNA-RNA nonhomologous recombination by utilizing local complementarity between recombining RNA molecules.
2. Brief Description of the Prior Art
Genetic recombination in RNA viruses is defined as any process involving the exchange of information between genomic RNA molecules. RNA recombination is of interest for several reasons. First, it offers a means for manipulating RNA genomes (including genetic mapping). Second, the ability to exchange genetic information may confer a selective advantage on the virus and thus be a significant factor in its evolution (1, 2).
Recombination processes, as defined above, are of two kinds: homologous and nonhomologous. In the former, the parent RNAs are related to each other and the location of the genetic crossover is the same in both sequences, so preserving any reading frame and producing a potentially functional recombinant molecule. In nonhomologous recombination neither of these restrictions applies.
We have studied recombination in brome mosaic virus (BMV) previously. Brome mosaic virus is a plant virus in which three species of genomic RNA have a similar, though not identical, sequence at the 3' end (3). RNA-RNA recombination in BMV was first demonstrated after coinoculation with a mixture of wild-type (wt) RNA1, wt RNA2, and a mutated RNA3 (designated M4). M4 contained a short deletion in the 3' noncoding sequence (4). The repaired RNA3 progeny resulted from crossovers between M4 RNA3 and either RNA1 or RNA2. In a majority of recombinants, the crosses occurred at homologous (legitimate) positions, while some contained duplications of 3' noncoding sequences. Characterization of a large number of recombinants suggested that BMV RNAs can form local heteroduplexes at the crossover sites (5, 6).
In addition to two bromoviruses, BMV and cowpea chlorotic mottle virus (CCMV) (7-10), RNA-RNA recombination has been demonstrated experimentally in other plant viruses (11, 12), in animal viruses (13-17), and in bacteriophages (18, 19). As in BMV, formation of local heteroduplexes was proposed to promote RNA-RNA recombination in poliovirus RNAs (14) or to be involved in generation of poliovirus defective interfering RNAs (15). Using poliovirus mutants to inhibit the replication of one parent, Kirkegaard and Baltimore (13) showed that recombination occurs via a copy-choice mechanism. In Sindbis virus, mutated RNAs induced illegitimate recombinants that contained both viral and non-viral insertions (17), most likely via a mechanism analogous to that observed in bromoviruses.
A discontinuous copy-choice mechanism has been proposed for recombination between genomic, defective interfering, and satellite RNAs of turnip crinkle virus (11, 20). The acceptor crossover sites appeared to corresponding to the recognition sequences of the turnip crinkle virus RNA replicase. Some form of discontinuous recombination mechanism also has been postulated for mouse hepatitis coronavirus (16). The crossover hotspots resulted from selection rather than from specific sequences (21), suggesting a random nature of recombination for mouse hepatitis coronavirus.
In the present invention, experimental evidence is provided for hybridization-mediated recombination in single stranded viruses. A construct formed from a first RNA molecule carrying an antisense insert complementary to a nucleotide sequence in a second target RNA molecule is used to direct crossovers between the first and second RNAs at or near the site of hybridization. The incidence of recombination and the location of recombinant junctions depends on the structure of the recombining RNA molecules and on the stability of the heteroduplex region.